Pseudomonas aeruginosa is one of the more important nosocomial pathogens, accounting for 10% of all hospital-acquired infections. The frequency and severity of P. aeruginosa infections underscore the need to understand better the pathogenic mechanism s utilized by this pathogen. Recently, a type III secretion system has been discovered in this organism and shown to play a role in disease. characterization of the system has already identified several interesting secreted toxins, and further examination is likely to find more. The hypothesis to be evaluated is that the P. aeruginosa type III system, like the type III system of several other gram-negative bacteria, secretes a factor that causes apoptosis of eukaryotic cells and plays a role in virulence. The PI proposes the following specific aims to evaluate this hypothesis: 1) Genetic and biochemical approaches will be utilized to identify the putative apoptosis-causing factors, which shall be referred to as ExoZ. 2) ExoZ will be introduced into eukaryotic cells to determine if it by itself is sufficient to cause apoptosis. 3) The role of ExoZ in disease will be investigated by comparing an isogenic mutant with the wild type parental strain in a mouse model of acute pneumonia. If initial attempts to identify ExoZ are not successful, the approaches used in specific aim 1 will be broadened to identify other cytotoxic factors, one of which will be chosen for in-depth analysis in specific aim 2 and 3. Successful completion of these specific aims will result in the identification and preliminary characterization of a P. aeruginosa type III secreted factor that causes apoptosis or is otherwise cytotoxic and the determination of this factor's role in disease. Such information will aid in the better understanding of P. aeruginosa pathogenesis and lead to novel therapeutic interventions. In addition, new insights gained from this work will lay the foundation for future investigations regarding the interaction of this factor with eukaryotic apoptosis cascades and the role of apoptosis in bacterial infections. Such advances are urgently needed given the increasing frequency of antibiotic resistance in P. aeruginosa clinical isolates.